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- PDB-6ee8: Mycobacterium tuberculosis RNAP promoter unwinding intermediate c... -

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Basic information

Entry
Database: PDB / ID: 6ee8
TitleMycobacterium tuberculosis RNAP promoter unwinding intermediate complex with RbpA/CarD and AP3 promoter
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (RNA polymerase-binding ...) x 2
  • DNA (60-MER)
  • DNA (65-MER)
  • RNA polymerase sigma factor SigA
KeywordsTRANSCRIPTION/DNA / initiation / half bubble / open promoter complex / TRANSCRIPTION / intermediate / TRANSCRIPTION-DNA complex
Function / homology
Function and homology information


bacterial-type RNA polymerase holo enzyme binding / response to water / bacterial-type RNA polymerase core enzyme binding / regulation of growth rate / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / stringent response / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase ...bacterial-type RNA polymerase holo enzyme binding / response to water / bacterial-type RNA polymerase core enzyme binding / regulation of growth rate / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / stringent response / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / cell wall / protein dimerization activity / transcription, DNA-templated / response to antibiotic / DNA-binding transcription factor activity / pathogenesis / positive regulation of transcription, DNA-templated / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytosol / cytoplasm
RNA polymerase-binding protein RbpA / RNA polymerase sigma factor RpoD, C-terminal / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase sigma factor RpoD ...RNA polymerase-binding protein RbpA / RNA polymerase sigma factor RpoD, C-terminal / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase sigma factor RpoD / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase sigma-70 region 1.2 / RNA polymerase beta subunit external 1 domain / CarD-like/TRCF domain superfamily / RNA polymerase, beta subunit, protrusion / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase Rpb2, domain 6 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 3 / CarD-like, C-terminal domain / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RbpA superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / Winged helix-like DNA-binding domain superfamily / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase, alpha subunit / CarD-like/TRCF domain / RNA polymerase-binding protein / Sigma-70, region 4 / Sigma-70 region 2 / Sigma-70 region 3 / Sigma-70 factor, region 1.2 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 1 / RNA polymerase sigma-70 / RNA polymerase Rpb1, domain 1 / RNA polymerase sigma-70 region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 region 3 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / CarD-like/TRCF domain / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 3 / RNA polymerase, N-terminal / RNA polymerase, subunit omega/K/RPB6 / DNA-directed RNA polymerase, omega subunit / RNA polymerase Rpb3/Rpb11 dimerisation domain / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Gyrase A; domain 2 / Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
DNA-directed RNA polymerase subunit beta / RNA polymerase-binding protein RbpA / RNA polymerase-binding transcription factor CarD / RNA polymerase-binding transcription factor CarD / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA ...DNA-directed RNA polymerase subunit beta / RNA polymerase-binding protein RbpA / RNA polymerase-binding transcription factor CarD / RNA polymerase-binding transcription factor CarD / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA / RNA polymerase-binding protein RbpA / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit alpha
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.92 Å
AuthorsDarst, S.A. / Campbell, E.A. / Boyaci Selcuk, H. / Chen, J.
CitationJournal: Nature / Year: 2019
Title: Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding.
Authors: Hande Boyaci / James Chen / Rolf Jansen / Seth A Darst / Elizabeth A Campbell /
Abstract: A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex. To generate the open complex, the conserved catalytic core of the RNAP combines ...A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12-14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed. Here we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2-universal structural features of RNAP-in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 13, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 21, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 23, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 30, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
F: RNA polymerase sigma factor SigA
J: RNA polymerase-binding protein RbpA
O: DNA (65-MER)
P: DNA (60-MER)
M: RNA polymerase-binding transcription factor CarD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)505,63913
Polymers505,48410
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE

#1: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 37745.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoA / Production host: Escherichia coli (E. coli)
References: UniProt: A5U8D3, UniProt: P9WGZ1*PLUS, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 125390.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoB, SAMEA2682864_01701 / Production host: Escherichia coli (E. coli)
References: UniProt: V9Z879, UniProt: P9WGY9*PLUS, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 148202.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoC / Production host: Escherichia coli (E. coli)
References: UniProt: A5U053, UniProt: P9WGY7*PLUS, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11776.996 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: rpoZ, ERS007672_03979, ERS007703_04032, ERS007720_04749, ERS027652_00548, ERS027654_02543, ERS027656_03959, ERS124361_02246
Production host: Escherichia coli (E. coli)
References: UniProt: A0A0T9N9K3, UniProt: P9WGY5*PLUS, DNA-directed RNA polymerase

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Protein , 1 types, 1 molecules F

#5: Protein RNA polymerase sigma factor SigA / Sigma-A


Mass: 58169.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: sigA, mysA, rpoD, rpoV / Production host: Escherichia coli (E. coli) / References: UniProt: P9WGI0, UniProt: P9WGI1*PLUS

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RNA polymerase-binding ... , 2 types, 2 molecules JM

#6: Protein RNA polymerase-binding protein RbpA


Mass: 12993.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rbpA / Production host: Escherichia coli (E. coli) / References: UniProt: P9WHJ4, UniProt: P9WHJ5*PLUS
#9: Protein RNA polymerase-binding transcription factor CarD


Mass: 17933.361 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: carD / Production host: Escherichia coli (E. coli) / References: UniProt: P9WJG2, UniProt: P9WJG3*PLUS

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DNA chain , 2 types, 2 molecules OP

#7: DNA chain DNA (65-MER)


Mass: 27907.809 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)
#8: DNA chain DNA (60-MER)


Mass: 27618.625 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)

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Non-polymers , 2 types, 3 molecules

#10: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#11: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mycobacterium tuberculosis RNAP promoter unwinding intermediate
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9 / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1.4 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 140333 / Symmetry type: POINT
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00730543
f_angle_d0.94841907
f_dihedral_angle_d13.48918224
f_chiral_restr0.0554775
f_plane_restr0.0075070

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